1. Field of the Invention
The present invention relates to uniformly sized polymeric microspheres, to methods of making the microspheres and to their use in labelling cell surfaces.
2. Description of the Prior Art
The isolation and characterization of cell membranes and their components is essential for an understanding of the role in which surface membranes play in regulating a wide variety of biological and immunological activities. The present techniques used for this purpose are unsatisfactory.
Most biological studies depend on the knowledge of cell membrane structure and its surface properties because cell functions are controlled by surface interactions between cell membranes and their environment. Thus the cell membrane properties determine the uptake of nutrients, growth, adhesion to other cells or artificial surfaces, removal of foreign particles (phagocytosis) etc. Of particular interest are the cell membrane receptors of normal and transformed cells and the interaction of specific sites on the membrane with chemotherapeutic or cytotoxic agents. Information on this interaction is necessary for practical applications, e.g. development of diagnostic tests or destruction of diseased cells. The separation of cell membranes and the isolation of cell receptors is the first step in the study of their properties or their interaction with natural or foreign elements. Although several methods of membrane separation are at present used and new methods are being investigated, none of them is satisfactory and a good technique for isolation of specific sites on the membrane surface is non-existent.
Knowledge of the nature, number and distribution of specific receptors on cell surfaces is of central importance for an understanding of the molecular basis underlying such biological phenomena as cell-cell recognition in development, cell communication and regulation by hormones and chemical transmitters, and differences in normal and tumor cell surfaces. In previous studies, the localization of antigens and carbohydrate residues on the surface of cells, notably red blood cells and lymphocytes, has been determined by bonding antibodies or lectins to such macromolecules as ferritin, hemocyanin or peroxidase which have served as markers for transmission electron microscopy. With advances in high resolution scanning electron microscopy (SEM), however, the topographical distribution of molecular receptors on the surfaces of cell and tissue specimens can be readily determined by similar histochemical techniques using newly developed markers resolvable by SEM.
Recently commercially available polystyrene latex particles have been utilized as immunologic markers for use in the SEM technique. The surface of such polystyrene particles is hydrophobic and hence certain types of macromolecules such as antibodies are adsorbed on the surface under carefully controlled conditions. However, such particles stick non-specifically to many surfaces and molecules and this seriously limits their broad application. Though these particles, while of the proper density for separating adsorbed or labelled membranes from other membranes are unchanged and are not capable of any derivitization by ionic or covalent bonding of protein and other biological molecules, and are thus limited in use to generalized studies on phagocytosis.
The preparation of small, stable, spherical particles which are bio-compatible, i.e., do not interact non-specifically with cells or other biological components and which contain functional groups to which specific proteins and other bio-chemical molecules can be covalently bonded is disclosed in copending application Ser. No. 434,124, filed Jan. 17, 1974, now U.S. Pat. No. 3,957,741 issued May 18, 1976.
The hydroxyl or amino groups can be activated by cyanogen bromide for covalent bonding of proteins and other chemicals containing amino groups to the polymeric latex. Methacrylic acid residues which impart a negative charge onto the particles are likely to prevent non-specific binding to cell surfaces and to provide carboxyl groups to which a variety of bio-chemical molecules can be covalently bonded using the carbodiimide method. Cross-linking of the polymeric matrix is essential to maintain the stability and size of the particles in both aqueous solution and in organic solvents commonly used in the fixation and dehydration of biological specimens for electron or light microscopy.
The polymeric microspheres ranging in diameter from 300 to 2000A have been successfully utilized as biocompatible immunochemical markers of red blood cells and lymphotcytes in scanning electric and light microscopy. However, the density of the microspheres is so close to that of the cell membranes that isolation of the cell membranes or bound receptor sites is not possible.